Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that is expressed at high levels on endothelial intercellular junctions and regulates transendothelial migration of leukocytes through unknown mechanisms. The investigators have recently observed that PECAM-1 engagement by antibodies results in increases in intracellular Ca++ levels in endothelial cells (EC). Since increases in Ca++ are important for many endothelial functions, including neutrophil migration across cell monolayers, the goals of this proposal are to study mechanisms how PECAM engagement regulates EC Ca++ influx and to directly test the hypothesis that PECAM mediated calcium regulation plays a role in neutrophil transmigration. To accomplish this, the investigators propose to 1) Determine the type of PECAM-1 engagement needed to trigger increase in intracellular Ca++, by engaging PECAM with a panel of epitope-mapped anti-PECAM antibodies, evaluating the role of Ab clustering, by treating cells with a soluble PECAM IgG chimera, and by using a cell-cell binding assay; 2) Define the regions in the cytoplasmic domain of PECAM required to increase Ca++, by transfecting a non-PECAM expressing cell line with a series of mutant PECAM molecules that contain truncations or mutations in the cytoplasmic domain, focusing first on the role of key tyrosine residues that undergo phosphorylation; 3) Determine mechanisms by which PECAM engagement leads to an increase in Ca++. Simultaneous patch clamping and intracellular calcium measurements in EC will define the electrophysiologic characteristics of the presumed current and allow dissection of the signaling processes interposed between engagement of PECAM and channel opening; and 4) Test the role of PECAM induced calcium signaling in an in vitro model of neutrophil transmigration in which neutrophils will migrate through monolayers of model cell lines transfected with wild type and mutant PECAM-1 cDNA's. The ability of PECAM-1 to enhance transmigration will be correlated with ability of each mutant cell line to support PECAM-mediated calcium increases. These studies will have a number of important implications for vascular biology including the identification of a potentially novel endothelial cell calcium influx pathway regulated by cell-cell interactions and a better understanding of the basic mechanisms of transendothelial leukocyte migration.